This invention relates to streptolysin O (SLO) antigens and their use, especially in diagnostic tests. In particular the present invention relates to the identification, construction and production of non-cytolytic and non-toxic SLO derivatives retaining antigenic sites that can detect antibodies in serum samples. The invention also relates to use of these SLO derivatives in diagnostic tests based on specific binding properties, such as binding between an antigen and an antibody.
SLO is a toxic cytolytic protein produced by Streptococcus pyogenes (S.pyogenes) which causes a number of human diseases. During infection, the gene encoding SLO is expressed and SLO is secreted by S. pyogenes. The toxicity of SLO seems to be closely associated with its cytolytic activity.
The infected human host produces anti-SLO antibodies to antigenic sites on the SLO molecule. Thus diagnostic tests detecting these anti-SLO antibodies in human serum, can indicate (past or present) infection by S.pyogenes. The immunodiagnostic assays presently being used for detection of anti-SLO antibodies in human serum utilise impure active SLO protein. These assays generally comprise the following steps:
(a) Take serum sample from patient. PA0 (b) Make serial dilutions of serum sample in a suitable buffer. PA0 (c) For each test include a control containing buffer, but no serum. PA0 (d) Add a standard quantity of active SLO to each dilution of serum and to the control. PA0 (e) Incubate the mixtures for a standard time and at a standard temperature, to allow any anti-SLO antibodies in the mixtures to combine with, and neutralise the added SLO. PA0 (f) Add a standard quantity of red blood cells to each mixture. PA0 (g) Incubate the mixtures for a standard time and at a standard temperature to allow any active (non-neutralised) SLO to lyse the added cells. PA0 (h) Determine the highest dilution of serum that has neutralised the added SLO, that is which corresponds to the dilution producing less than 50% lysis of the added red blood cells.
Thus, where the serum sample contains high levels of anti-SLO antibody, there will be neutralisation of the active SLO up to a high dilution of the serum and therefore no lysis of the red blood cells. Conversely, where the serum sample contains lower levels of antibodies to SLO, there will be neutralisation only at low dilutions of the sample and there will be extensive lysis of the red blood cells at high dilutions of the sample.
However, there are a number of problems with these assays. The assays are difficult, time-consuming and require laboratory facilities. They utilise active SLO and, as stated, this protein is toxic and cytolytic and therefore laboratory facilities and trained personnel are required. The preparation of purified SLO from S. pyogenes is difficult and costly, particularly as SLO is sensitive to degradation by proteases produced by the S. pyogenes.
The present assays for detecting anti-SLO antibodies also use impure preparations of SLO which are unstable in liquid form. Thus, the SLO preparations are supplied as lyophilized powder in vials, each vial containing sufficient material for a set number of serum tests. Before use, the lyophilized powder must be reconstituted in a suitable solvent. However, the reconstituted SLO rapidly loses its activity, probably owing to the presence of contaminating proteases, and therefore it must either be used within a short time or discarded. Thus, it is costly to test individual serum samples as soon as they arrive in the laboratory. To overcome this problem laboratories generally store the samples until they have a sufficient number to enable economic use of a vial of lyophilized SLO. This means that there may be up to one weeks delay between taking the serum sample and obtaining the test result.